ROR1 plays a critical role in pancreatic tumor-initiating cells with a partial EMT signature

  1. Masaya Yamazaki
  2. Shinjiro Hino
  3. Shingo Usuki
  4. Yoshihiro Miyazaki
  5. Tatsuya Oda
  6. Mitsuyoshi Nakao
  7. Takaaki Ito
  8. Kazuya Yamagata

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Abstract

Tumor-initiating cells are the major drivers of chemoresistance and relapse, making them attractive targets for cancer therapy. However, the identity of tumor- initiating cells in human pancreatic ductal adenocarcinoma (PDAC) and the key molecules underlying their traits remain poorly understood. Here, we show that a partial epithelial-mesenchymal transition (EMT)-like subpopulation marked by high expression of receptor tyrosine kinase-like orphan receptor 1 (ROR1) is the origin of heterogeneous tumor cells in PDAC. We demonstrate that ROR1 depletion suppresses tumor growth, recurrence after chemotherapy, and metastasis. Mechanistically, ROR1 induces the expression of AURKB by activating E2F to enhance PDAC proliferation. Furthermore, epigenomic analyses reveal that ROR1 is transcriptionally dependent on YAP/BRD4 binding at the enhancer region, and targeting this pathway reduces ROR1 expression and prevents PDAC growth. Collectively, our findings reveal a critical role of ROR1 high cells as tumor-initiating cells and the functional importance of ROR1 in PDAC progression, thereby highlighting its therapeutic targetability.

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    Reply to the reviewers

    1. General Statements

    We thank the reviewer for stating that “The detailed analysis uses many state of the art techniques to address the role of ROR1 and is of great interest to a large audience including basic researchers in the field of cancer biology and oncologists in the clinic” and we appreciate the reviewer’s constructive suggestions. We have substantially revised our manuscript and plan to perform new experiments based on these valuable comments.

    1. Description of the planned revisions

    Three main points: (1) The importance of AURKB as a downstream effector of ROR1 [Reviewer #1: major #2] Based on these suggestions, we plan to perform a colony formation assay using AURKB-overexpressing cells with ROR1-knockdown. We will clarify this point in the revised manuscript.

    (2) The link between ROR1 expression and YAP/BRD4 [Reviewer #1: major #5 and Reviewer #3: major #1] Based on the suggestion, we plan to perform the luciferase reporter assay. We will clearly describe this experiment in the revised manuscript.

    (3) Single-cell analysis using other models to validate tumor heterogeneity [Reviewer #2: major #1 and Reviewer #3: major #2] Based on your suggestion, we plan to analyze primary human tumors (public data: for example, GSE155698, CRA001160) and examine PDO#1 xenografts (in-house data). We will clearly state this information in the revised manuscript.

    For the two minor points suggested by Reviewer #2, we plan to (1) reanalyze TCGA data. (2) perform the organoid or colony formation assay to validate that the siRNA model functionally recapitulates the ROR1low vs. ROR1high phenotype.

    Please see the “Authors’ responses to the reviewers' comments” for more details.

    1. Description of the revisions that have already been incorporated in the transferred manuscript

    As suggested by the reviewer, we have substantially revised our manuscript, and the changes are shown in red. • Reviewer #1: major comments #2, #3, #4, and #5; minor comments #1 and #2 • Reviewer #2: major comments #2, #3, and #4; minor comments #2, #3, #4, #8, and #10 • Reviewer #3: minor comments #1 and #2

    Please see the “Authors’ responses to the reviewers' comments” for more details.

    1. Description of analyses that authors prefer not to carry out

    Authors’ responses to the reviewers' comments

    Reviewer #1

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    In this manuscript the authors analyzed the role of ROR1 in pancreatic cancer progression and metastasis. They found that ROR1 expression is specifically increased in an partial EMT cell cluster upon scRNA-Seq of tumor cells derived from an orthotopic mouse PDAC model. Moreover, the ROR1 high population in tumors specifies cells with high proliferation and tumor initiation capacities, increased metastatic propensity and chemoresistance, since knockdown of ROR1 shows reduction of these features in vivo. By comparing transcriptomes from several in vivo models the authors identified that ROR1 acts through AURKB and that its expression is regulated by an upstream enhancer that is bound by YAP/TAZ and BRD4 complexes. With this study the authors identified a new targetable pathway that promotes tumor progression and metastasis in PDAC. The detailed analysis uses many state of the art techniques to address the role of ROR1 and is of great interest to a large audience including basic researchers in the field of cancer biology and oncologists in the clinic. However, some of the findings are a bit preliminary and the drawn conclusions are not sufficiently supported by the experimental data. Moreover, some findings seem a bit out of context and do not really help to bring the story forward. At other instances experimental details are missing to mechanistically demonstrate the role of ROR1. In particular it remains elusive how ROR1 is regulated, i.e. which signaling events are crucial to generate ROR1 high vs. low cells. I listed my specific comments below.

    [Response] We thank the reviewer for stating that “The detailed analysis uses many state of the art techniques to address the role of ROR1 and is of great interest to a large audience including basic researchers in the field of cancer biology and oncologists in the clinic” and we appreciate the reviewer’s constructive suggestions. We have substantially revised our manuscript and plan to perform new experiments based on these valuable comments.

    1. The authors' initial finding is that in the partial EMT cluster ROR1, but also other RTKs (out of 56) are specifically increased. What about the other RTKs? Why was ROR1 chosen to analyze more thoroughly?

    [Response 1] We are thankful for the reviewer’s suggestion to clarify why ROR1 was selected. (1) Seven candidate genes (EPHA4, EPHA7, ERBB4, FGFR1, JAK3, LYN, and ROR1) were chosen as surface markers in the partial EMT cluster. (2) The genes were sorted in order of high expression. (3) ROR1 is reported to promote metastasis in breast cancer (Cui et al, 2013). The induction of metastasis is one of the functions of tumor-initiating cells. FGFR1 is already known to enhance the CSC-like phenotype in non-small cell lung cancer (Ji et al, 2016). (4) The antibody against ROR1 was marketed as available for cell sorting using FACS. Therefore, we focused on ROR1 as a potential new marker for tumor-initiating cells with a partial EMT signature.

    References Cui B, Zhang S, Chen L, Yu J, Widhopf GF 2nd, Fecteau JF, Rassenti LZ, Kipps TJ. Targeting ROR1 inhibits epithelial-mesenchymal transition and metastasis. Cancer Res. 2013 Jun 15;73(12):3649-60. doi: 10.1158/0008-5472.CAN-12-3832. PMID: 23771907; PMCID: PMC3832210. Ji W, Yu Y, Li Z, Wang G, Li F, Xia W, Lu S. FGFR1 promotes the stem cell-like phenotype of FGFR1-amplified non-small cell lung cancer cells through the Hedgehog pathway. Oncotarget. 2016 Mar 22;7(12):15118-34. doi: 10.18632/oncotarget.7701. PMID: 26936993; PMCID: PMC4924774.

    1. The finding of AURKB as crucial target of ROR1 is very weak and needs more in-depth analyses. It is not clear why AURKB was chosen over the other candidates. Is AURKB expression directly regulated by ROR1? Are the two genes directly linked? Can ROR1 deficiency be compensated by AURKB overexpression? Especially the decrease in AURKB protein level in Fig. 4K is not very convincing to account for the different phenotypes in ROR1 high and low cells. Is AURKB and ROR1 expression correlated in TCGA samples (like Fig. 8B)? In Fig. 4L the readout was changed from colony numbers to colony diameter. If AURKB is the crucial player downstream of ROR1, then colony formation efficiency should be affected at first. This needs to be shown. The statement in lines 223,224 that AURKB is a direct downstream target of ROR1 was not shown!

    [Response 2-1: changed] We thank the reviewer for noting this issue. We have performed additional experiments to assess the hypothesis that AURKB is a crucial downstream target of ROR1. ROR1-knockdown not only suppressed AKT phosphorylation (Supplemental Figure 9A) but also decreased c-Myc protein levels and the expression of c-Myc target genes (CDK4, CCND1, CDK2, and CCNE1), leading to a reduction in RB phosphorylation (new Supplemental Figure 9B and 9C). Based on these results, ROR1 regulates c-Myc expression through AKT signaling, leading to the activation of the E2F network (new Supplemental Figure 9D). We added some figures and descriptions to the preliminary revision manuscript (new Supplemental Figure 9B–9D, lines 357–363, lines 649–651).

    [Response 2-2: the planned revisions] We also plan to perform new experiments with a colony formation assay to determine whether ROR1 deficiency is compensated by AURKB overexpression. We agree that this experiment will confirm that AURKB is an important downstream target of ROR1 in PDAC proliferation.

    [Response 2-3] In TCGA-PAAD dataset, AURKB expression was not correlated with ROR1 expression. Since the ROR1high cluster is a minor population in the tumor, a downstream analysis of specific clusters with results from a bulk study such as this TCGA dataset is difficult to perform.

    [Response 2-4: changed] We have added a new graph of organoid formation efficiency (new Figure 4L) and changed some descriptions in the preliminary revision manuscript (line 227).

    1. Fig. 4 A-E: The ROR1 KD was induced in vitro but not continued in vitro. The transient KD has a strong impact on tumor forming capacity, even though recovery of expression is likely within the first days in vivo. This is very interesting and underscores the role of ROR1 in tumor initiation and presumably independent of differences in proliferation. Would the results be different, if the DOX treatment would start with injection of the cells and continued in vivo? Is then tumor initiation not affected and maybe only tumor growth?

    [Response 3: changed] We apologize for the confusing description in the original manuscript. In Fig. 4A–E, we used PDAC cells with stable expression of doxycycline-inducible shROR1. ROR1-knockdown was maintained in vivo by adding doxycycline to the drinking water. Continuous ROR1-knockdown suppressed tumor growth (Fig. 4C–E). Several statements we made were more ambiguous than intended, and we have adjusted the text and the figures for clarity in the preliminary revision manuscript (new Figure 4A and B, lines 203–204).

    1. In Fig. 5 the authors show that ROR1 is highly expressed in tumors after gemcitabine treatment and conclude that the ROR1 high cells are a resistant population. However, this statement is too strong, since gemcitabine treatment could also lead to an upregulation of ROR1 in "low" cells during acquisition of chemoresistence. Together with our knowledge on the role of EMT in driving therapy resistance and therapy-mediated induction of EMT, such a scenario is equally likely. Similarly, the statement in lines 370-372 is not supported by experimental evidence.

    [Response 4: changed] We appreciate the reviewer’s critical comments. As suggested, we have not clearly determined whether (1) the ROR1high cells survived gemcitabine treatment and/or (2) the ROR1low cells increased ROR1 expression upon exposure to this treatment. We have carefully changed some descriptions in the preliminary revision manuscript (lines 241–242, 382–383).

    1. In order to understand how ROR1 is regulated, the authors use ATAC-Seq and cut and run and identified a putative upstream enhancer element (Fig. 7). Although this element increases the activity of the promoter fragment in a reporter construct, the experiments do not help to understand how ROR1 activity is increased specifically in the "high" cells. Are peaks of YAP1 and BRD4 also changed between hi/lo cells? Is YAP OE and KD (BRD4 OE and KD) or the use of the inhibotor JQ1 altering the activity of the reporter constructs (i.e. only of the enhancer-promoter combination but not of the promoter only construct)? This would help to strengthen a direct link between ROR1, YAP and BRD4. Is YAP activity different in ROR1 high vs. low cells?

    [Response 5-1: changed] We thank the reviewer for this important comment. We have shown differences in chromatin accessibility and histone modification of the ROR1 enhancer between ROR1high and ROR1low cells using ATAC-seq and CUT&RUN assays (Fig. 7B). Very few ROR1high/low cells are present in xenograft. We were not successful in experiments examining the binding of YAP and BRD4 to enhancers in ROR1high/low cells because of the technical limitations in the ChIP and CUT&RUN assays. Instead, we used public data to examine YAP and BRD4 occupancy at the ROR1 enhancer region of cell lines with low ROR1 expression. In T-47D and MCF7 cells (breast cancer cells, low ROR1 expression), YAP and BRD4 did not bind to the ROR1 enhancer region (new Figure 8D and 8I). We have added figures and some descriptions to the preliminary revision manuscript (new Figure 8D and 8I, lines 304–309, line 768).

    [Response 5-2: the planned revisions] We plan to perform new experiments with the reporter assay you suggested. We agree that this experiment will help strengthen the direct link between ROR1, YAP and BRD4.

    [Response 5-3] As shown in Figure 8C, GSEA revealed that ROR1high cells in both S2-VP10 xenografts and PDO#1 xenografts expressed higher levels of YAP-regulated genes than ROR1low cells in these xenografts. We have added a description of this result as follows: “Thus, ROR1high cells have higher YAP activity than ROR1low cells.” (lines 304–305).

    1. In Fig. 8A the authors identified 202 antigens that match the H3 monomethylation / acetylation pattern. How was YAP etc. chosen?

    [Response 6] We apologize for the poor description in the original manuscript. We chose YAP and BRD4 based on the following criteria: (1) these antigens are expressed in S2-VP10 cells and PDO#1 and (2) bind to the ROR1 enhancer region (based on an analysis of public data).

    Minor:

    1. Fig. 2D,E: What is actually shown here? Is there an overlap between the genes that define ROR1 high vs. low cells in both approaches? The gene list should be provided.

    [Response: changed] We apologize for the poor description in the original manuscript. We have added this information to the preliminary revision manuscript (new Supplemental Table 3).

    1. Fig. 3G: I suggest to include the images of the tumors from the ROR1 low cells in the main figure as well.

    [Response: changed] We appreciate the reviewer’s suggestion. We have moved this information from the supplementary information to the main figure in the preliminary revision manuscript (new Figure 3G, lines 186–189).

    Reviewer #1 (Significance (Required)):

    PDAC is a very aggressive desease with very low 5-year survival rates. Understanding of the pathobiology is of keen interest. The findings of the authors are of high significance and extremely relevant as they provide a mechanism that can also be targeted by specific drug combinations, i.e. standard care gemcitabine with specific ROR1 inhibition. The findings are of great interest to a large audience including basic researchers in the field of cancer biology and oncologists in the clinic.

    [Response] We greatly appreciate the reviewer’s comments.

    Reviewer #2

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    In this work Yamazaki and colleagues performed single cell RNA sequencing of one xenograft tumor formed by the S2-VP10 PDAC cell line to explore PDAC intratumor heterogeneity. Using this model they identified ROR1 as heterogeneously expressed in neoplastic cells. Using further in vivo and in vitro models they show that ROR1high cells have higher tumor initiation capacity than ROR1low. By histone and ATAC-seq analyses, they identify a ROR1 enhancer upstream the promoter and show that YAP and BRD4 bind to this genomic region and that BRD4 inhibition by JQ1 reduces ROR1 expression and organoid formation. The data, figures and methods are nicely and clearly presented.

    [Response] We thank the reviewer for stating that “The data, figures and methods are nicely and clearly presented”, and we appreciate the reviewer’s constructive suggestions. We have substantially revised our manuscript and plan to perform new experiments based on these valuable comments.

    Major comments

    1. The authors use one xenograft tumor as starting model and all conclusions are derived from the data generated with this model. To support the existence of identifie heterogeneity in the PDAC neoplastic compartment, I would strongly suggest to validate the existence of the partial EMT population and the ROR1 heterogeneity in single cell data bases generated from primary human tumors.

    [Response 1: the planned revisions] We thank the reviewer for the positive suggestion. We plan to perform a new analysis of available public single-cell data from human PDAC tumors. In addition, we also launched a single-cell analysis of PDO#1 xenografts.

    1. In Fig. 3G, it is mentioned that tumors grown from ROR1high cells recapitulate the original PDOx histology thus suggesting that ROR1high cells in the tumor are the actual TICs. ROR1low cells could also grow tumors, just with lower incidence. Are these tumors any different to the ROR1high derived ones? Is it just a lower tumor initiation capacity (TIC) or they can not recapitulate the tumor as the ROR1high cell? Can they also give rise to differentiated progeny cells? This should appear in the main text and not only in the discussion. I would suggest to move panel 3G to supplementary figure.

    [Response 2: changed] We thank the reviewer for noting this issue and apologize for the confusing description in the original manuscript. ROR1low cells generated tumors at a low frequency, and these tumors showed a hierarchical histology mimicking the original tumor. As suggested, we have added this information to the main text (new Figure 3G, lines 186–189).

    1. In line 160 you mention that known CSC markers such as CD44, PROM1 and DCLK1 are not differentially expressed between ROR1 high and low populations. Then, in figure 3H,I you analyze the expression of CD44v6 together with ROR1. I would try to put this information together in the text, or at least in fig. 3 start with something like "we had seen that both ROR1high and low express CD44, however...". In any case, I feel that the experiment with CD44 could be obviated (or at least moved to supplementary), as it brings the question of weather this is also true for DCLK1 or CD133.

    [Response 3: changed] We appreciate and agree with the reviewer's comment on this point. Accordingly, we have moved this figure to the supplementary information and changed the description (new Supplemental Figure 5C and 5D, lines 191–196).

    1. JQ1 has been described to inhibit PDAC growth by downregulation of MYC. To unequivocally link the effect of JQ1 in the downregulation of ROR1 (Fig. 8M) as discussed in the text it would be important to exclude that other mechanisms such as MYC downregulation are taking place. For example, does JQ1 treatment of ROR1low cells also reduce their colony formation capacity (in an experiment such as the one in fig. 3C). Or does ROR1 re-expression in Fig. 8M rescue the JQ1 effect? These or other experiments could help to establish a stronger link between (BRD4/JQ1) and ROR1.

    [Response 4: changed] We thank the reviewer for this important comment. As mentioned in the response to Reviewer #1-major comment #2, we newly found that ROR1 regulates c-Myc expression through AKT signaling, leading to the activation of the E2F network (new Supplemental Figures 9B–9D, lines 357–363).

    Minor comments

    1. The data are nicely presented (text and figures) and the conclusions are clear. My suggestion to make the story more "catchy" at the beginning would be, if possible, to start from the observation done in primary human data and then move to the PDX model to explore ROR1 as a TIC marker in PDAC. For this, you could use available public single cell data of human PDAC tumors. If this doesn't work (it is of course possible that by unsupervised analysis you don't get the same clusters as in the PDX with the partial EMT cluster popping up), it would be nice if some primary tumor data came early in the story (currently the first figure showing heterogeneity in primary samples is in supplem fig. 4A).

    [Response: the planned revisions] We thank the reviewer for these excellent comments. As suggested, we plan to perform several new analyses (please see the previous comment for details: Reviewer #2-major comment #1).

    1. It is not clear if the xenografts were subcutaneous or orthotopic. It would be good to include this information in the main text (line 102) and the methods so that the reader knows what is the exact model that has been used.

    [Response: changed] We thank the reviewer for this comment and apologize for the poor description in the original manuscript. As suggested, we have added this information to the preliminary revision manuscript (line 101).

    1. In Fig. 2F and 2G I would highlight the EMT pathway to help the reader.

    [Response: changed] We thank the reviewer for this comment. As suggested, we have changed the relevant figures in the preliminary revision manuscript (new Figure 2F and 2G).

    1. In Supp Fig 4B it would be nice to have an amplified view of the staining as in panel C of the same figure.

    [Response: changed] We thank the reviewer for this comment. As suggested, we have added high-magnification images of the staining in the preliminary revision manuscript (new Supplemental Figure 4A and 4B).

    1. In the same figure (Fig. 4A-D) ROR1 shows an apical staining pattern that doesn't seem to resemble the staining in patient samples. I am not an expert in pathology evaluation but I would recommend a pathologist to give her/his opinion. Possibly, during the PDX process, few cells from the original patient tumor are selected giving a different staining pattern.

    [Response] We appreciate the reviewer's comment on this point. Dr. Ito, a coauthor of this paper, is a pathologist. We have changed some images of staining in patient samples (new Supplemental Figure 4A). We agree that ROR1 shows an apical staining pattern in PDX samples. However, some sites show similar apical staining patterns in patient samples (Patient #2 and Patient #4 in the new Supplemental Figure 4A). We propose that PDX mimics the original patient tissue because it has heterogeneity of ROR1 expression and morphological features indicative of a luminal structure.

    1. In the analyses of TCGA data, be aware that only 150 from the original dataset are actual PDAC tumors. The dataset contains otherwise data from cell lines, PDX, normal tissue, etc that should be removed for a proper analysis (see DOI: 10.3390/cancers11010126)

    [Response: the planned revisions] We thank the reviewer for the careful review of this issue. We are currently reconsidering with the pathologist whether the samples are appropriate based on TCGA data (diagnosis and pathology sections) and the paper you presented. The current data (Figures 3A, 4J, and 8B) were analyzed for samples excluding cell lines, PDX, and normal tissue in the TCGA-PAAD dataset.

    1. Does ROR1 correlate with RFS? This would nicely fit with the concept of TIC and metastasis.

    [Response] We thank the reviewer for noting this issue. Unfortunately, no correlation was observed between ROR1 expression and RFS.

    1. Line 219: ROR1 is not "depleted" in the lines as it is a downregulation model. "ROR1-downregulated" would be more correct.

    [Response: changed] We thank the reviewer for this suggestion and agree with your comment. We have corrected this term accordingly in the preliminary revision manuscript (line 223).

    1. It would be good to have a supplem figure showing that siROR1 cells show reduction organoid formation, to validate that the siRNA model functionally recapitulates the ROR1low vs high phenotype.

    [Response: the planned revisions] We thank the reviewer for this suggestion. We plan to perform a colony formation assay.

    1. Some of the supplemental figures are only referred in the discussion although they appear earlier than other in the main text. This is a bit confusing when going through the figures.

    [Response] We apologize for the poor description in the original manuscript. We have adjusted the order of the supplemental figures in the preliminary revision manuscript.

    CROSS-CONSULTATION COMMENTS I agree with the importance of addressing points 2 (link to AURKB), 4 (selection vs acquisition), 5 (mechanism in high vs low cells) raised by Reviewer 1, and the comments from Reviewer 3. I think that the study of other RTKs (point 1 from Reviewer 1) is not the focus of the story. It would be nice if the authors can comment on why they chose ROR1 but the fact that are other differentially expressed genes does not exclude the validity of the current story. I fell that the in vivo sustained KD experiment (point 3 from Reviewer 1) although interesting, it is not mandatory for a revision of this manuscript in case the adaptation of the animal protocol represents a long process. The experiment provided already in the current version is the best approach to address the role of ROR1 at the early initiation phase.

    [Response] We thank the reviewer for these positive comments. As suggested, we have substantially revised our manuscript.

    Reviewer #2 (Significance (Required)):

    Significance: This is a neat and interesting work with potential implications for the clinical field of pancreatic cancer as the authors identified a new subpopulation with enhanced tumor initiating cell capacity. However, the use of JQ1 for pancreatic cancer has been previously discussed mainly linked to MYC inhibition, but also to stromal reprogramming or DNA damage induction. I missed some discussion in this regard in the discussion section. What is adding the work to the field of JQ1 treatment in PDAC? IN a way, how do the authors foresee that the discovery of ROR1high cells and the regulation of ROR1 by BRD4 and YAP will be beneficial when considering JQ1 in the clinics? Maybe by stratifying patients? Or by following ROR1 upregulation upon initial chemotherapy? These questions are just suggestions. In general, some discussion to put the work into the context of previous works using JQ1 in PDAC would be nice.

    [Response: changed] We thank the reviewer for this comment. As you suggested, we have added a description of the proposed use of JQ1 and BRD4 inhibitors in ROR1high PDAC treatment to the Discussion section (lines 412–416).

    I believe that this work would be interesting not only to the pancreatic cancer community but also to a more general public working on cancer and/or stemmness as it touches several interesting points in that regard that can be applicable to other systems. My own work is focused on pancreatic cancer, patient heterogeneity and stromal interactions. I am not an expert on histone or ATACseq analyses.

    [Response] We greatly appreciate the reviewer’s comments.

    Reviewer #3

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Summary Yamazaki et al investigate partial EMT in pancreatic cancer and provide data that ROR1 marks pancreatic tumor cells that are capable of initiating tumors. The authors exploit scRNAseq of pancreatic tumor xenografts to identify a cluster of cells showing a partial EMT phenotype. The found 7 RTKs expressed more highly in this partial EMT cluster and focus their attention on ROR1, an 'orphan' receptor that has been implicated in WNT signaling and EMT previously. Validation experiments using ROR1-high vs low cells support that ROR1 expression correlates with EMT, poor outcome in human PDA patients, tumor forming and colony forming capacity. They also show that ROR1 high cells form tumors that recapitulate parental tumor histology. The authors show that ROR1 expression is associated with EF2 transcription factor activity, elevated expression of multiple targets including AURKB. Pharmacologic inhibition of AURKB reduces colony formation and genetic loss of ROR1 combined with chemotherapy (gemcitabine) has potent anti-tumor activity in vivo. The authors show that ROR1 expression is elevated in metastatic lesions and identify a novel enhancer element that putatively drives ROR1 expression in tumor cells. They provide evidence that this element is engaged by YAP/BRD4 and show that BRD4 inhibition reduces tumor cell colony formation. The manuscript is a solid combination of techniques with adequate controls and statistics.

    [Response] We thank the reviewer for stating that “The manuscript is a solid combination of techniques with adequate controls and statistics”, and we appreciate the reviewer’s constructive suggestions. We have substantially revised our manuscript and plan to perform new experiments based on these valuable comments.

    Major Comments: The overall conclusion that ROR1 expression marks a subset of pancreatic cancer cells that have the ability to initiate tumors is supported by the data provided. The correlative data are strong and the demonstration that loss of ROR1 reduces colony formation, reduces metastatic lesions and enhances the efficacy of chemotherapy are compelling. Additionally, the demonstration that ROR1 expression is elevated in metastatic lesions is consistent with many other drivers/markers of EMT in pancreatic cancer.

    The conclusion that ROR1 expression is driven by YAP/BRD4 is interesting and provides important mechanistic depth to the study. However, this conclusion could be strengthened by use of a suitable rescue experiment. For instance does overexpression of ROR1 rescue the effect of BRD4 inhibition or loss of YAP?

    [Response 1: the planned revisions] We thank the reviewer for this comment. We completely agree with the reviewer’s suggestion. However, the suggested examination to determine whether overexpression of ROR1 rescues the effect of BRD4 inhibition or loss of YAP may not be suitable because BRD4 and YAP act as transcriptional coregulators of various target genes. Instead, as mentioned in response to Reviewer #1-major comments 5-2, we plan to perform new experiments using a reporter assay.

    A challenge with the data presented in Figure 1, the scRNA-seq data that lead them to ROR1, is that it is not stated how many tumors are used to generate the scRNA-seq data and the overall number of tumor cells analyzed is relatively low (993). The authors should provide the number of tumors used for the initial scRNA-seq. A general concern with any scRNA-seq data is batch effect, this is mitigated to a degree by the follow on studies that provide functional validation of ROR1 in multiple cell lines.

    [Response 2: changed and the planned revisions] We appreciate the reviewer’s comments. As suggested, we have added this information to the preliminary revision manuscript (line 104). In addition, as mentioned in response to Reviewer #2 major comment #1, we plan to perform a new single-cell analysis of PDO xenografts (in-house data) and human PDAC tumors (available public data).

    The data and methods are provided in an adequate manner. Reproduction of the experiments is likely. The authors use multiple cell lines and tools that are generally available. The authors note a limitation of the study is that only human tumor xenografts were exploited.

    [Response] We thank the reviewer for the positive comment.

    Minor comments: Figure 1E and text page 9. The text identifies MERB3 as a gene that marks the partial EMT cluster, I believe this is a type and the gene should actually be MSRB3.

    [Response: changed] We apologize for the typo. We have corrected this error accordingly (line 114).

    Please provide the dose of gemcitabine in the legend for figure 5

    [Response: changed] We apologize for the poor description in the original manuscript. We have added this information.

    CROSS-CONSULTATION COMMENTS I think the comments from Referee #2 are pretty reasonable - have no additions

    Reviewer #3 (Significance (Required)):

    Intratumor heterogeneity is a major challenge for the treatment of many cancers, including pancreatic cancer. The data provided support that ROR1 marks a subset of cancer cells in pancreatic tumors that have the capacity to drive intratumor heterogeneity. If supported these data have the potential to drive significant impact. Identification of a marker and a targetable pathway that supports tumor initiation in pancreatic cancer has the potential to nominate companion therapies that enhance the efficacy of standard of care approaches. Further, identification of a pathway that drives partial EMT in pancreatic cancer provides a substantial increase in baseline knowledge of intratumor heterogeneity.

    These data would be broadly interesting to scientists interested in the tumor microenvironment, metastasis, therapy resistance and tumor progression. In addition, oncologists focused on drug development and combinatorial therapy will find this manuscript of interest.

    [Response] We greatly appreciate the reviewer’s comments.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary

    Yamazaki et al investigate partial EMT in pancreatic cancer and provide data that ROR1 marks pancreatic tumor cells that are capable of initiating tumors. The authors exploit scRNAseq of pancreatic tumor xenografts to identify a cluster of cells showing a partial EMT phenotype. The found 7 RTKs expressed more highly in this partial EMT cluster and focus their attention on ROR1, an 'orphan' receptor that has been implicated in WNT signaling and EMT previously. Validation experiments using ROR1-high vs low cells support that ROR1 expression correlates with EMT, poor outcome in human PDA patients, tumor forming and colony forming capacity. They also show that ROR1 high cells form tumors that recapitulate parental tumor histology. The authors show that ROR1 expression is associated with EF2 transcription factor activity, elevated expression of multiple targets including AURKB. Pharmacologic inhibition of AURKB reduces colony formation and genetic loss of ROR1 combined with chemotherapy (gemcitabine) has potent anti-tumor activity in vivo. The authors show that ROR1 expression is elevated in metastatic lesions and identify a novel enhancer element that putatively drives ROR1 expression in tumor cells. They provide evidence that this element is engaged by YAP/BRD4 and show that BRD4 inhibition reduces tumor cell colony formation. The manuscript is a solid combination of techniques with adequate controls and statistics.

    Major Comments:

    The overall conclusion that ROR1 expression marks a subset of pancreatic cancer cells that have the ability to initiate tumors is supported by the data provided. The correlative data are strong and the demonstration that loss of ROR1 reduces colony formation, reduces metastatic lesions and enhances the efficacy of chemotherapy are compelling. Additionally, the demonstration that ROR1 expression is elevated in metastatic lesions is consistent with many other drivers/markers of EMT in pancreatic cancer.

    The conclusion that ROR1 expression is driven by YAP/BRD4 is interesting and provides important mechanistic depth to the study. However, this conclusion could be strengthened by use of a suitable rescue experiment. For instance does overexpression of ROR1 rescue the effect of BRD4 inhibition or loss of YAP?

    A challenge with the data presented in Figure 1, the scRNA-seq data that lead them to ROR1, is that it is not stated how many tumors are used to generate the scRNA-seq data and the overall number of tumor cells analyzed is relatively low (993). The authors should provide the number of tumors used for the initial scRNA-seq. A general concern with any scRNA-seq data is batch effect, this is mitigated to a degree by the follow on studies that provide functional validation of ROR1 in multiple cell lines.

    The data and methods are provided in an adequate manner. Reproduction of the experiments is likely. The authors use multiple cell lines and tools that are generally available.

    The authors note a limitation of the study is that only human tumor xenografts were exploited.

    Minor comments:

    Figure 1E and text page 9. The text identifies MERB3 as a gene that marks the partial EMT cluster, I believe this is a type and the gene should actually be MSRB3.

    Please provide the dose of gemcitabine in the legend for figure 5

    Referees cross-commenting

    I think the comments from Referee #2 are pretty reasonable - have no additions

    Significance

    Intratumor heterogeneity is a major challenge for the treatment of many cancers, including pancreatic cancer. The data provided support that ROR1 marks a subset of cancer cells in pancreatic tumors that have the capacity to drive intratumor heterogeneity. If supported these data have the potential to drive significant impact. Identification of a marker and a targetable pathway that supports tumor initiation in pancreatic cancer has the potential to nominate companion therapies that enhance the efficacy of standard of care approaches. Further, identification of a pathway that drives partial EMT in pancreatic cancer provides a substantial increase in baseline knowledge of intratumor heterogeneity.

    These data would be broadly interesting to scientists interested in the tumor microenvironment, metastasis, therapy resistance and tumor progression. In addition, oncologists focused on drug development and combinatorial therapy will find this manuscript of interest.

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    Referee #2

    Evidence, reproducibility and clarity

    In this work Yamazaki and colleagues performed single cell RNA sequencing of one xenograft tumor formed by the S2-VP10 PDAC cell line to explore PDAC intratumor heterogeneity. Using this model they identified ROR1 as heterogeneously expressed in neoplastic cells. Using further in vivo and in vitro models they show that ROR1high cells have higher tumor initiation capacity than ROR1low. By histone and ATAC-seq analyses, they identify a ROR1 enhancer upstream the promoter and show that YAP and BRD4 bind to this genomic region and that BRD4 inhibition by JQ1 reduces ROR1 expression and organoid formation. The data, figures and methods are nicely and clearly presented.

    Major comments

    1. The authors use one xenograft tumor as starting model and all conclusions are derived from the data generated with this model. To support the existence of identifie heterogeneity in the PDAC neoplastic compartment, I would strongly suggest to validate the existence of the partial EMT population and the ROR1 heterogeneity in single cell data bases generated from primary human tumors.
    2. In Fig. 3G, it is mentioned that tumors grown from ROR1high cells recapitulate the original PDOx histology thus suggesting that ROR1high cells in the tumor are the actual TICs. ROR1low cells could also grow tumors, just with lower incidence. Are these tumors any different to the ROR1high derived ones? Is it just a lower tumor initiation capacity (TIC) or they can not recapitulate the tumor as the ROR1high cell? Can they also give rise to differentiated progeny cells? This should appear in the main text and not only in the discussion. I would suggest to move panel 3G to supplementary figure.
    3. In line 160 you mention that known CSC markers such as CD44, PROM1 and DCLK1 are not differentially expressed between ROR1 high and low populations. Then, in figure 3H,I you analyze the expression of CD44v6 together with ROR1. I would try to put this information together in the text, or at least in fig. 3 start with something like "we had seen that both ROR1high and low express CD44, however...". In any case, I feel that the experiment with CD44 could be obviated (or at least moved to supplementary), as it brings the question of weather this is also true for DCLK1 or CD133.
    4. JQ1 has been described to inhibit PDAC growth by downregulation of MYC. To unequivocally link the effect of JQ1 in the downregulation of ROR1 (Fig. 8M) as discussed in the text it would be important to exclude that other mechanisms such as MYC downregulation are taking place. For example, does JQ1 treatment of ROR1low cells also reduce their colony formation capacity (in an experiment such as the one in fig. 3C). Or does ROR1 re-expression in Fig. 8M rescue the JQ1 effect? These or other experiments could help to establish a stronger link between (BRD4/JQ1) and ROR1.

    Minor comments

    1. The data are nicely presented (text and figures) and the conclusions are clear. My suggestion to make the story more "catchy" at the beginning would be, if possible, to start from the observation done in primary human data and then move to the PDX model to explore ROR1 as a TIC marker in PDAC. For this, you could use available public single cell data of human PDAC tumors. If this doesn't work (it is of course possible that by unsupervised analysis you don't get the same clusters as in the PDX with the partial EMT cluster popping up), it would be nice if some primary tumor data came early in the story (currently the first figure showing heterogeneity in primary samples is in supplem fig. 4A).
    2. It is not clear if the xenografts were subcutaneous or orthotopic. It would be good to include this information in the main text (line 102) and the methods so that the reader knows what is the exact model that has been used.
    3. In Fig. 2F and 2G I would highlight the EMT pathway to help the reader.
    4. In Supp Fig 4B it would be nice to have an amplified view of the staining as in panel C of the same figure.
    5. In the same figure (Fig. 4A-D) ROR1 shows an apical staining pattern that doesn't seem to resemble the staining in patient samples. I am not an expert in pathology evaluation but I would recommend a pathologist to give her/his opinion. Possibly, during the PDX process, few cells from the original patient tumor are selected giving a different staining pattern.
    6. In the analyses of TCGA data, be aware that only 150 from the original dataset are actual PDAC tumors. The dataset contains otherwise data from cell lines, PDX, normal tissue, etc that should be removed for a proper analysis (see DOI: 10.3390/cancers11010126)
    7. Does ROR1 correlate with RFS? This would nicely fit with the concept of TIC and metastasis.
    8. Line 219: ROR1 is not "depleted" in the lines as it is a downregulation model. "ROR1-downregulated" would be more correct.
    9. It would be good to have a supplem figure showing that siROR1 cells show reduction organoid formation, to validate that the siRNA model functionally recapitulates the ROR1low vs high phenotype.
    10. Some of the supplemental figures are only referred in the discussion although they appear earlier than other in the main text. This is a bit confusing when going through the figures.

    Referees cross-commenting

    I agree with the importance of addressing points 2 (link to AURKB), 4 (selection vs acquisition), 5 (mechanism in high vs low cells) raised by Reviewer 1, and the comments from Reviewer 3. I think that the study of other RTKs (point 1 from Reviewer 1) is not the focus of the story. It would be nice if the authors can comment on why they chose ROR1 but the fact that are other differentially expressed genes does not exclude the validity of the current story. I fell that the in vivo sustained KD experiment (point 3 from Reviewer 1) although interesting, it is not mandatory for a revision of this manuscript in case the adaptation of the animal protocol represents a long process. The experiment provided already in the current version is the best approach to address the role of ROR1 at the early initiation phase.

    Significance

    This is a neat and interesting work with potential implications for the clinical field of pancreatic cancer as the authors identified a new subpopulation with enhanced tumor initiating cell capacity. However, the use of JQ1 for pancreatic cancer has been previously discussed mainly linked to MYC inhibition, but also to stromal reprogramming or DNA damage induction. I missed some discussion in this regard in the discussion section. What is adding the work to the field of JQ1 treatment in PDAC? IN a way, how do the authors foresee that the discovery of ROR1high cells and the regulation of ROR1 by BRD4 and YAP will be beneficial when considering JQ1 in the clinics? Maybe by stratifying patients? Or by following ROR1 upregulation upon initial chemotherapy? These questions are just suggestions. In general, some discussion to put the work into the context of previous works using JQ1 in PDAC would be nice.

    I believe that this work would be interesting not only to the pancreatic cancer community but also to a more general public working on cancer and/or stemmness as it touches several interesting points in that regard that can be applicable to other systems.

    My own work is focused on pancreatic cancer, patient heterogeneity and stromal interactions. I am not an expert on histone or ATACseq analyses.

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript the authors analyzed the role of ROR1 in pancreatic cancer progression and metastasis. They found that ROR1 expression is specifically increased in an partial EMT cell cluster upon scRNA-Seq of tumor cells derived from an orthotopic mouse PDAC model. Moreover, the ROR1 high population in tumors specifies cells with high proliferation and tumor initiation capacities, increased metastatic propensity and chemoresistance, since knockdown of ROR1 shows reduction of these features in vivo. By comparing transcriptomes from several in vivo models the authors identified that ROR1 acts through AURKB and that its expression is regulated by an upstream enhancer that is bound by YAP/TAZ and BRD4 complexes. With this study the authors identified a new targetable pathway that promotes tumor progression and metastasis in PDAC. The detailed analysis uses many state of the art techniques to address the role of ROR1 and is of great interest to a large audience including basic researchers in the field of cancer biology and oncologists in the clinic. However, some of the findings are a bit preliminary and the drawn conclusions are not sufficiently supported by the experimental data. Moreover, some findings seem a bit out of context and do not really help to bring the story forward. At other instances experimental details are missing to mechanistically demonstrate the role of ROR1. In particular it remains elusive how ROR1 is regulated, i.e. which signaling events are crucial to generate ROR1 high vs. low cells. I listed my specific comments below.

    1. The authors' initial finding is that in the partial EMT cluster ROR1, but also other RTKs (out of 56) are specifically increased. What about the other RTKs? Why was ROR1 chosen to analyze more thoroughly?
    2. The finding of AURKB as crucial target of ROR1 is very weak and needs more in-depth analyses. It is not clear why AURKB was chosen over the other candidates. Is AURKB expression directly regulated by ROR1? Are the two genes directly linked? Can ROR1 deficiency be compensated by AURKB overexpression? Especially the decrease in AURKB protein level in Fig. 4K is not very convincing to account for the different phenotypes in ROR1 high and low cells. Is AURKB and ROR1 expression correlated in TCGA samples (like Fig. 8B)? In Fig. 4L the readout was changed from colony numbers to colony diameter. If AURKB is the crucial player downstream of ROR1, then colony formation efficiency should be affected at first. This needs to be shown. The statement in lines 223,224 that AURKB is a direct downstream target of ROR1 was not shown!
    3. Fig. 4 A-E: The ROR1 KD was induced in vitro but not continued in vitro. The transient KD has a strong impact on tumor forming capacity, even though recovery of expression is likely within the first days in vivo. This is very interesting and underscores the role of ROR1 in tumor initiation and presumably independent of differences in proliferation. Would the results be different, if the DOX treatment would start with injection of the cells and continued in vivo? Is then tumor initiation not affected and maybe only tumor growth?
    4. In Fig. 5 the authors show that ROR1 is highly expressed in tumors after gemcitabine treatment and conclude that the ROR1 high cells are a resistant population. However, this statement is too strong, since gemcitabine treatment could also lead to an upregulation of ROR1 in "low" cells during acquisition of chemoresistence. Together with our knowledge on the role of EMT in driving therapy resistance and therapy-mediated induction of EMT, such a scenario is equally likely. Similarly, the statement in lines 370-372 is not supported by experimental evidence.
    5. In order to understand how ROR1 is regulated, the authors use ATAC-Seq and cut and run and identified a putative upstream enhancer element (Fig. 7). Although this element increases the activity of the promoter fragment in a reporter construct, the experiments do not help to understand how ROR1 activity is increased specifically in the "high" cells. Are peaks of YAP1 and BRD4 also changed between hi/lo cells? Is YAP OE and KD (BRD4 OE and KD) or the use of the inhibotor JQ1 altering the activity of the reporter constructs (i.e. only of the enhancer-promoter combination but not of the promoter only construct)? This would help to strengthen a direct link between ROR1, YAP and BRD4. Is YAP activity different in ROR1 high vs. low cells?
    6. In Fig. 8A the authors identified 202 antigens that match the H3 monomethylation/acetylation pattern. How was YAP etc. chosen?

    Minor:

    1. Fig. 2D,E: What is actually shown here? Is there an overlap between the genes that define ROR1 high vs. low cells in both approaches? The gene list should be provided.
    2. Fig. 3G: I suggest to include the images of the tumors from the ROR1 low cells in the main figure as well

    Significance

    PDAC is a very aggressive desease with very low 5-year survival rates. Understanding of the pathobiology is of keen interest. The findings of the authors are of high significance and extremely relevant as they provide a mechanism that can also be targeted by specific drug combinations, i.e. standard care gemcitabine with specific ROR1 inhibition. The findings are of great interest to a large audience including basic researchers in the field of cancer biology and oncologists in the clinic.

  5. Version published to 10.1101/2022.07.13.499868v1 on bioRxiv